Iron trafficking and metabolism in macrophages: contribution to the polarized phenotype

Iron trafficking and metabolism in macrophages: contribution to the polarized phenotype

Opinion Iron trafficking and metabolism in macrophages: contribution to the polarized phenotype Gaetano Cairo1, Stefania Recalcati1, Alberto Mantovan...

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Opinion

Iron trafficking and metabolism in macrophages: contribution to the polarized phenotype Gaetano Cairo1, Stefania Recalcati1, Alberto Mantovani2,3 and Massimo Locati2,3 1

Department of Human Morphology and Biomedical Sciences ‘‘Citta` Studi’’, University of Milan, Milan, Italy Department of Translational Medicine, University of Milan, Milan, Italy 3 IRCCS Istituto Clinico Humanitas, Rozzano, Milan, Italy 2

During inflammation, proinflammatory macrophages sequester iron as a well known bacteriostatic mechanism. Alternative activation of macrophages is linked to tissue repair, and during this process the expression pattern of genes important for iron homeostasis is distinct from that in proinflammatory macrophages. This leads to an increased capacity of the alternatively activated macrophages for heme uptake, via scavenger receptors, and for production of anti-inflammatory mediators via heme-oxygenase-dependent heme catabolism. Alternatively activated macrophages also release non-heme iron into tissues via ferroportin. Here, we propose that the iron-release-associated phenotype of alternatively activated macrophages significantly contributes to their role in various conditions, including tissue repair and tumor growth. Macrophage activation and polarization The concept of polarized macrophage activation emerged several years ago as a framework to describe the diverse functions displayed by this highly plastic cell type under different conditions [1]. It is now established that, in response to different signals from the microenvironment, macrophages can adopt a continuum of different functional profiles. This can range from classical (or M1) activation, which is typically observed in inflammatory conditions and in response to pathogen challenge, to the other extreme of alternative (or M2) activation, which is driven by immune signals including interleukin (IL)-4, IL-13, transforming growth factor b and glucocorticoids. M2 macrophages are characteristic of the resolution phase of inflammation. These distinct activation phenotypes support different, and in some cases, opposing biological functions, which have an impact on tissue homeostasis and various pathological conditions, including infectious diseases and cancer [2–7]. An understanding of macrophage polarization has been guided by knowledge of how lymphocytes are polarized during adaptive immune activation; thus, a major focus has been the immune regulators that contribute to polarization. Cytokines and chemokines, in particular, have been best proven to distinguish M1 (IL-12high IL-10low) and M2 (IL-12low IL-10high) macrophages [3,6]. Recent studies have Corresponding author: Cairo, G. ([email protected]).

drawn attention to the relevance of the macrophage metabolic profile during activation [8]. Gene expression profiles related to iron metabolism are strikingly polarized between different types of activated macrophages, with >60% of genes associated with iron homeostasis by GeneOntology being differentially expressed between human M1 and M2 macrophages [10]. Here, we briefly discuss iron trafficking during the ‘classical’ response of inflammatory macrophages, and then consider iron trafficking in M2 macrophages, with a focus on heme-associated iron. We also explore the implications of this ‘alternative’ regulation of iron availability under the physiological conditions, and pathological processes in which M2 macrophages are involved. Iron metabolism in inflammatory macrophages Iron is an essential growth factor for most bacteria and parasites, which have evolved multiple mechanisms to acquire iron from environments where little free iron is available (Box 1). Conversely, iron retention in the reticuloendothelial system is a well characterized response to inflammation, and is generally viewed as an attempt by the host to withhold iron from invading pathogens (for further insights, see [11,12]). The host counteracts microbial strategies employed to maximize iron uptake mostly through proinflammatory signals that act on macrophages, which increase the macrophage uptake of heme-associated iron (consisting of an iron atom contained in a porphyrin ring) and non-heme-associated iron [13] and decreased expression of ferroportin (Fpn), which is the only known cellular exporter of non-heme-associated iron [14–17]. In addition, infected macrophages synthesize and secrete lipocalin-2, which prevents siderophore-mediated iron capture by pathogens [16], and also rapidly recruit natural resistance-associated macrophage protein 1 (NRAMP1) to the phagosomal membrane to export iron into the cytoplasm [18]. These mechanisms deplete the phagosomes of iron and are essential for successful resistance to infection, because iron overload owing to iron supplementation or erythrocyte transfusion results in exacerbation of the disease [13]. This is illustrated by in vitro results that have shown that growth of Mycobacterium tuberculosis is impaired in macrophages of patients with hereditary hemochromatosis [19] who have inappropriate, low levels

1471-4906/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2011.03.007 Trends in Immunology, June 2011, Vol. 32, No. 6

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Box 1. How host and pathogens compete for various forms of iron The complexity and redundancy of pathogen mechanisms to acquire iron indicates the importance of this poorly available yet essential metal, which is used for DNA synthesis, respiration, and free radical detoxification [11,12]. Most bacteria secrete siderophores (high-affinity iron-binding organic molecules), which are bound by specific iron-regulated receptors on the bacterial outer membrane. After internalization, the complex reaches the cytoplasm where iron is liberated. Some highly adapted pathogens can also use host heme and non-heme iron proteins as iron sources (Figure 1). S. aureus induces hemolysis and then imports hemoglobin-derived heme and uses heme oxygenase-like proteins to liberate iron [81]. Other pathogens, such as Neisseria, have evolved the ability to bind host iron-containing proteins (hemoglobin, transferrin, and lactoferrin), to transport iron into their periplasmic space. Iron acquisition represents a special challenge for intracellular pathogens; in particular, those that reside within the endocytic pathway of mammalian cells, because endosomes and lysosomes are gradually depleted in iron by host transporters. Salmonella, Mycobacteria, and the protozoon Leishmania have evolved efficient countermeasures, such as iron transporter systems that effectively compete with host transporters for iron, thus indicating that a pool of intraphagosomal iron is crucial for their intracellular survival and

[()TD$FIG]

replication. To use better different types of siderophores for outstripping iron from transferrin in the endosome [82], M. tuberculosis blocks the maturation of phagosomes and fusion with lysosomes, to maintain this intracellular niche at an early endosomal stage [11]. Moreover, M. tuberculosis induce Mramp [83], a homolog of the mammalian NRAMP1 that pumps iron out of the phagosome [84], to move iron in the opposite direction. The molecular strategy used by Leishmania for survival in the iron-poor environment of macrophage phagolysosomes involves activation of Tf receptor 1 expression, to exploit the major iron uptake system of the host cell [85], and the induction of LIT1, a ZIP family membrane Fe2+ transporter [86]. In spite of their efficient uptake mechanisms, microorganisms might have iron deficiency. In that case, they transcriptionally activate the so-called iron regulon to enhance iron uptake capacity [87], and exploit novel post-transcriptional control mechanisms to repress specifically gene expression and distribute iron only to essential iron-containing proteins (reviewed in [88]). As bacteria increasingly become resistant to antibiotics, advances in our understanding of the nutritional needs of the microorganisms with respect to iron, will provide useful guidelines for the development of alternative antimicrobial intervention strategies based on the limitation of iron availability (Figure I).

Lipocalin 2

24p3R

Siderophore-bound Fe

Fe-siderophore importers DMT1 Host

Fe Importers

Free Fe

Pathogen Transferrin receptor 1 Lactoferrin receptor

Protein-bound iron (transferrin, lactoferrin)

Outer membrane receptors Heme transport system

HCP1 CD91

CD163

Heme iron Hemopexin Haptoglobin-hemoglobin

Hemoglobin TRENDS in Immunology

Figure I. Pathogen-host competition for different forms of extracellular iron. Mammals and microorganisms have evolved a variety of molecules to bind different forms of iron, with the dual purpose of acquiring this essential metal for their own necessities and, at the same time, to limit availability for the opponent. The control of this strategic resource is an important element of the host-pathogen interaction and of the so-called nutritional immunity.

of iron in macrophages [20], as well as in vivo evidence that indicates that mice with hemochromatosis (Hfe–/–) have an improved control of Salmonella typhimurium infection and significantly better survival [21]. To avoid the possibility that defense mechanisms to starve extracellular microorganisms of iron could facilitate the growth of intracellular bacteria [17], macrophages also limit iron available to intracellular bacteria [15,16] via interferon 242

(IFN)g-dependent mechanisms [14]. Finally, it is particularly interesting to mention that the effects exerted by local proinflammatory mediators are integrated with systemic mechanisms that also have an impact on iron metabolism (Box 2). In summary, under inflammatory conditions, local and systemic pathways increase iron uptake and repress iron export mechanisms, which leads to iron sequestration in inflammatory macrophages.

Opinion Box 2. Impact of the systemic inflammatory response on macrophage iron metabolism During the acute phase of systemic inflammation, inflammatory cytokines induce the liver to secrete the iron-binding proteins Tf and lactoferrin, and this represents a well-known primary defense mechanism. Liver hepcidin production is also induced, which inhibits macrophage iron release by targeting Fpn to degradation [89]. These events lead to iron sequestration in activated macrophages at the site of inflammation/infection to restrict iron availability to the pathogens locally. Inflammatory cytokines in the circulation also induce iron retention in spleen macrophages and also in the Kupffer cells that recycle great amounts of iron from effete erythrocytes. Therefore, these reticuloendothelial cells are probably the major cell type responsible for the hypoferremia that occurs during systemic infection. Incidentally, the chronic sequestration response might restrict iron availability for erythroid precursors, and hence, contribute towards causing inflammationrelated anemia; a common condition in patients with infections, tumors, and autoimmune disorders [90]. During a systemic inflammatory response, proinflammatory cytokines also activate a classical neuroendocrine–immune negative regulatory loop via the hypothalamic–pituitary–adrenal axis, which results in adrenal production of endogenous glucocorticoids [91]. Macrophage exposure to glucocorticoids induces an alternatively activated phenotype, which is characterized by high CD163 expression and enhanced hemoglobin clearance and detoxification capability, suppressed Tf receptor and high Fpn expression [40]. Thus, iron metabolism in macrophages is strongly influenced by polarizing agents also during the development of a systemic inflammatory response, with circulating proinflammatory cytokines that sustain an iron-retention phenotype, and eventually, the development of inflammation-related anemia, and endogenous glucocorticoids that support a global shift of iron homeostasis toward an increased cellular export of heme-derived iron. It is tempting to speculate that, because iron retention by inflammatory macrophages contributes to pathogen control during the acute phase of inflammation, the ability of alternatively activated macrophages to donate iron could provide a relevant contribution to tissue repair in the resolution phase.

Heme uptake and catabolism in alternatively activated macrophages A distinctive property of M2 macrophages is the elevated expression of scavenger receptors, which contribute to inflammation resolution by clearing surrounding tissue of cell debris and modified molecules, such as oxidized lipoproteins [7]. Data suggest that M2 macrophages also play a role in heme scavenging, in that CD163, whose synthesis is controlled by several cytokines that sustain M2 polarization, mediates internalization of the haptoglobin–hemoglobin complex [22–25], and CD91 mediates the uptake of hemopexin-bound heme [26]. Moreover, the heme carrier protein HCP-1, which might co-localize with CD163 in the early endosomes of macrophages, is inhibited by IFNg produced in response to lipopolysaccharide (LPS), and is upregulated by the glucocorticoid dexamethasone [27]. HCP-1 is also a folate transporter (proton-coupled folate transporter) [28] that is involved in folate-receptormediated endocytosis [29]. The folate receptor appears to be a marker for anti-inflammatory macrophages [30]. Scavenging of heme is a relevant mechanism to prevent the toxic properties of extracellular hemoglobin that is derived from the hemolysis that accompanies inflammation [31] and severe sepsis [32], and to limit the pro-oxidant and proinflammatory activity of heme, which is released from hemoglobin [33] or degraded hemoproteins such as catalase, cytochromes, peroxidases, and myeloperoxidases that

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are derived from neutrophils. Overall, this clearing response could be beneficial by limiting the inflammatory reaction. Also to be considered is that the increased heme pool in M2 macrophages inhibits the binding of the transcription repressor Bach1 to specific sequences in heme oxygenase 1 (HO-1) promoter, thus allowing the transcription activator NF-E2-related factor 2 to bind and activate HO-1 expression [34]. HO-1 activation, a characteristic of the M2 metabolic signature, occurs not only in response to increased heme availability, but also in response to M2 polarizing agents, including IL-10 [35]. HO-1 catalyzes the rate limiting step in the oxidative degradation of heme into three byproducts: biliverdin, which is subsequently converted to the antioxidant and anti-inflammatory bilirubin, ‘free’ ferrous iron, and CO [36]. CO is a toxic gas that also has signaling properties and has been shown to have beneficial effects in a number of conditions, including the inflammatory response [36,37]. In conclusion, HO-1 counteracts inflammation and thus can contribute to the biological properties of alternative macrophages. It is tempting to speculate that the elevated capacity for heme uptake in M2 macrophages might also represent a pathway to provide substrate to HO-1, thus allowing this enzyme to exert its protective and anti-inflammatory effects [38,39]. Iron recycling in alternatively activated macrophages The protective role of HO-1 presents an apparent paradox considering that HO-1 liberates metabolically active ferrous iron, which could potentially catalyze oxidative reactions, but ferritin, which is usually concomitantly activated, chelates iron in a non-toxic form [36]. In contrast to other cell types, however, the increased availability of heme-associated and non-heme-associated iron in M2 macrophages does not trigger ferritin synthesis. Instead, Fpn expression is upregulated [10,24,25,40]. The release of free iron derived from heme catabolism in the cytosolic labile iron pool inhibits the activity of the iron regulatory proteins that translationally regulate Fpn [41], thus allowing efficient Fpn translation. Additionally, heme activates Fpn transcription [42,43] through a Maf recognition element in the Fpn promoter [44]. Both regulatory mechanisms cooperate to increase Fpn expression on the cell membrane, thus leading to increased ferrous iron export [10]. That M2 macrophages do not downregulate [10,24], or only marginally increase [40] ferritin in response to the iron load derived from heme degradation is in line with the iron recycling role of macrophages. Iron recycling might depend on high basal Fpn expression, which is also directly induced at the transcriptional level by M2-polarizing signals, including glucocorticoids [27] and IL-4 [10]. The elevated levels of Fpn might drive the flux of iron from cytosolic storage into ferritin toward Fpn at the plasma membrane, which consequently favors iron release. Recent studies that have shown concomitant HO-1 induction and Fpn-mediated iron release in cultured M2 macrophages [10,24] are in line with studies showing that HO-1 knockdown in mice results in reticuloendothelial iron retention that eventually leads to damage and death of splenic and liver macrophages [45,46], as well as with previous 243

Opinion indications that HO-1 favors iron release [47]. It is also possible that some heme escapes breakdown and is exported via the recently identified heme export protein FLVCR, which is highly expressed in macrophages [48]. Finally, it should be noted that the macrophage phenotype is dynamically modulated by the microenvironment, which can promote the switch from the M1 to the M2 state. Therefore, it is also conceivable that the M2 macrophages might release the iron previously accumulated under the influence of proinflammatory stimuli. Pathophysiological implications As mentioned, iron retention in M1 macrophages is key in bacteriostasis [11,12], and anemia during chronic disease has been clearly defined (Box 2). Conversely, the pathophysiological implications of heme uptake and degradation, and subsequent iron release by M2 macrophages are largely unknown. Based on the fragmentary evidence available, a number of scenarios can be proposed. Effects of low intracellular iron availability Low intracellular iron is associated with reduced nuclear factor (NF)-kB activity in endothelial cells, in which decreased iron availability impairs the expression of adhesion molecules [49], inhibits translation of proinflammatory cytokines tumor necrosis factor a and IL-6, and reduces LPS-induced cytokine expression in macrophages [50,51]. Macrophage iron deficiency might also favor the activation of hypoxia inducible factors (HIFs) because iron is required for constitutive HIF degradation [52]; in particular, a relative iron deficiency might increase the activity of HIF-2, which is induced during an M2 response [53], and the expression of its target genes (e.g. arg1) in M2 macrophages. In addition, Fpn-dependent iron depletion seems to decrease inducible nitric oxide synthase (iNOS) expression at the translational level [54]; however, because iron chelation increases iNOS transcription [55] in macrophages in an HIF-1 [56] and NF-IL6 [57] -dependent manner, the role of iron availability in iNOS expression remains unclear. Iron scarcity in M2 macrophages could also prevent the formation of functional iron-containing enzymes that are involved in arachidonate metabolism, such as lipoxygenases and prostaglandin H synthase, or myeloperoxidase; thus blunting the proinflammatory response. Also affected could be synthesis of the iron-containing enzyme tyrosine hydroxylase (TH), which catalyzes the rate-limiting step in catecholamine biosynthesis. It has been shown that LPS-stimulated macrophages activate TH and release catecholamines, which leads to an enhanced inflammatory response and tissue damage [58,59] that is characterized by activation of NF-kB and macrophage proinflammatory cytokine production [60]. An impaired TH-mediated response in M2 macrophages might be relevant for downmodulating the inflammation to prevent tissue injury; in particular, during infectious disease when catecholamines cause transferrin (Tf) and lactoferrin iron release, thus providing iron for siderophores and favoring bacterial growth [61], as exemplified by the ability of catecholamine inotropic drugs to stimulate iron-dependent staphylococcal proliferation and biofilm formation [62]. Finally, iron availability might also influence acquired immune 244

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functions, because M2 macrophages are no longer able to activate T cells under conditions of iron deficiency [24]. In summary, low intracellular iron levels could represent a key element in the anti-inflammatory phenotype of M2 macrophages. Effects of high extracellular iron availability M2 macrophages have been demonstrated to export actively non-heme-associated iron [10,24]. The functional implications of this activity are largely undefined, but it is reasonable to assume that iron released as Fe2+ by Fpn might be bound by Tf and internalized by Tf-receptor-1mediated endocytosis by adjacent cells. The metal could be then used for a variety of purposes, because iron-containing proteins are essential for oxygen transport, cell respiration, DNA replication and cell growth, for example [10]. This might represent a process that is aimed at resolving inflammation and allowing repair, consistent with the documented relevance of iron release from M2 macrophages in muscle repair [22,24]. More generally, increased iron availability in the extracellular milieu could influence the growth rate of adjacent fibroblasts and extracellular matrix deposition (prolyl hydroxylases, which are required for correct collagen biosynthesis, also are iron-dependent enzymes) during the repair phase and possibly during fibrosis [63–65]. Considering the toxicity of heme, proteins that bind, transport and degrade heme, in particular HO-1 [66], have been proposed to form a key system in the control of wound healing [67]. In our opinion, the export of iron derived from heme degradation could play an important role in this context. In line with this view, a recent study has shown that iron accumulation in macrophages obtained from human chronic venous leg ulcers contributes to the induction of a M1 phenotype characterized by production of reactive oxygen and nitrogen species that damage adjacent fibroblasts. Eventually, this results in an impaired capacity for tissue repair and chronic wounds that do not heal [68]. This study thus supplies proof of the concept of the different roles of M1 and M2 macrophages in wound healing as a function of iron availability. It can be hypothesized that the high Fpn-dependent iron release found in M2 cells might also occur in tumor-associated macrophages (TAMs), which are closely related to M2 macrophages [7]. We recently have shown that the conditioned medium of M2 macrophages sustains faster growth of malignant and non-malignant cell lines [10]. Therefore, in an in vivo situation of tumor growth, a high level of iron release might provide an unrestricted source of iron to cancer cells, which generally proliferate more rapidly than normal cells and hence require more iron. In line with this finding, HO-1 and CD163 coexpression has been recently shown in TAMs [25]. Increasing iron bioavailability in the tumor microenvironment might thus represent a previously unknown mechanism that underlies the tumor-promoting activity of TAMs, which are educated by tumors to release iron to fuel cancer cell proliferation. The importance of the Fpn-mediated control of iron availability in the tumor microenvironment is also supported by the reduced Fpn expression (and hence higher iron content) that has been recently found in breast cancer cells compared to normal breast epithelial cells [69]. Tumor Fpn levels in

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human breast cancer patients are inversely correlated with malignant potential and clinical outcome. Altogether, it emerges that iron and iron-related gene expression should be regarded as functional components of the tumor environment [70]. Effect of heme catabolism As mentioned, HO-1 activity degrades heme into CO, which promotes vasodilation and angiogenesis via several mechanisms, including HIF-1a-mediated induction of vascular endothelial growth factor [71], a well known M2 product [9]. It has also been demonstrated that CO suppresses Toll-like receptor 4 signaling [72] and blocks the LPS-mediated induction of proinflammatory cytokines by modulation of the mitogen-activated protein kinase pathway [73]. Thus, HO-1 on the one hand mediates the degradation of free and protein-bound heme, and on the other hand, promotes the formation of protective compounds. Moreover, one can imagine that CO, which specifically targets biologically relevant transition metals, such as manganese, iron, cobalt and copper that are present in structural and functional proteins, could interact with, and thus inactivate, the heme moiety of inflammatory hemoproteins, including iNOS, indoleamine 2,3-dioxygenase, cyclooxygenase (COX), prostaglandin H synthase, and NADPH oxidase [74]. Inhibition of membrane NADPH oxidase and subsequent downregulation of superoxide production are implicated in the anti-inflammatory effects of CO [75]. Finally, HO-1-dependent heme catabolism could result in a lack of intracellular heme and thus prevent the [()TD$FIG]synthesis of functional hemoproteins, as has been demonM1 macrophages (LPS, IFNγ)

strated for proteins involved in the inflammatory response, such as gp91 phox [76] and COX [77]. This HO-1-mediated effect is expected to affect preferentially fast turnover hemoproteins (COX-2 and iNOS), whereas constitutive hemoproteins (COX-1, endothelial NOS, and mitochondrial and microsomal proteins) would be spared [74]. In this respect, it might be relevant that, although M1 activation is associated with a strong induction in COX-2 expression, M2 activation results in a marked induction of COX-1 [9]. Pathogens such as Leishmania use the effect of HO-1dependent heme catabolism to prevent the synthesis of functional hemoproteins as a means to subvert macrophage polarization [76]. Concluding remarks Macrophage polarization is associated with differential expression of proteins that are involved in iron uptake, storage and release, with M2 macrophages displaying a phenotype associated with iron release, which is clearly different from the iron sequestration response induced under proinflammatory conditions in M1 macrophages [10,24,25,40]. This leads to differences in both intra- and extracellular iron availability in polarized macrophages and their microenvironment. The amount of heme-degrading enzyme HO-1 is elevated in M2 macrophages, which leads to the production of various anti-inflammatory mediators (Figure 1). As discussed, differential iron management by macrophages might have an impact on neighboring cells. A better understanding of iron metabolic pathways in polarized macrophages, as well as further characterization of M2 macrophages (IL-4) Heme uptake (CD163, CD91)

Heme uptake (CD163, CD91)

Heme pool

Heme pool Bilirubin

HO-1

Bilirubin Biliverdin

HO-1

CO

Biliverdin CO

Fe Fe

Iron storage (ferritin)

Iron exit (ferroportin)

Iron storage (ferritin)

Iron exit (ferroportin)

Iron retention

Bactericidal activity Proinflammatory cytokines Immunostimulation Bacteriostatic activity Tumour suppression

Iron release CO production

Matrix remodeling Angiogenesis Immunoregulation Tissue repair Tumour promotion TRENDS in Immunology

Figure 1. Iron metabolism in polarized macrophages. Macrophage polarization affects the expression of ferritin, which stores intracellular iron, and Fpn, which exports iron from the cell. M1 macrophages are characterized by a ferritinhigh/Fpnlow phenotype, which sustains their propensity to store iron and deplete the microenvironment. Conversely, M2 macrophages are characterized by a ferritinlow/Fpnhigh phenotype, and elevated levels of scavenger receptors and HO-1, which mediate efficient heme uptake and degradation, respectively. This metabolic profile sustains the propensity of M2 macrophages to take up and degrade heme and recirculate free iron to the microenvironment.

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Opinion macrophage subsets, might be relevant to the pathophysiology of various diseases. For example, the role of macrophage polarization in atherosclerotic plaques is not fully understood, and characterization of plaque-associated macrophages remains incomplete [78]. Advanced atheromatous lesions contain more iron than healthy arteries, and heme and iron are associated with lesion development and destabilization. The precise role of iron in atherosclerosis development is an open question. We anticipate that definition of macrophage iron trafficking in atherosclerotic lesions will provide important insights into disease pathogenesis, as suggested by the demonstration that the iron content of plaque-associated macrophages is important for atherosclerosis disease progress [79], and that HO-1 expression in macrophages has an antiatherogenic effect and a beneficial role by decreasing the inflammatory component of atherosclerosis [80]. Conflict of interest The authors declare no financial or commercial conflict of interest.

Acknowledgments The authors’ research activities in this field are supported by grants from the Italian Association for Cancer Research (AIRC), the Alleanza Contro il Cancro, the Istituto Superiore di Sanita` (Programma Straordinario di Ricerca Oncologica 2006-RNBIO project), the Italian Ministry for University and Research (MIUR; FIRB and PRIN projects), the Italian Ministry of Health, and Regione Lombardia (LIIN project).

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